The present invention relates to methods for the early detection of complications in pregnancy based on the detection of bioactive lipids. Specifically, the present invention relates to methods for early detection of preeclampsia and related disorders by detecting levels of glycerophosphatidyl compounds and lysophospholipids in a sample specimen obtained from a pregnant woman.
Preeclampsia is a condition responsible for up to 50-70% of hypertensive. complications in pregnancies. Although a leading cause of morbidity and mortality in pregnancies, its etiology and causes remain largely unknown. In severe cases, preeclampsia may develop into eclampsia, which often leads to death. Despite the dangers associated with preeclampsia, no cure exists for preeclampsia, although early detection and diagnosis enables therapy and treatment protocols that offer the best chance to save the lives of the baby and the mother.
Preeclampsia generally occurs after the 20th week of pregnancy and appears without much warning. The most common symptoms are high blood pressure, swelling or edema of hands and feet, and increased protein in the urine. In the United States, preeclampsia is responsible for up to 10% of pregnancy-related mortality and morbidity each year. Preeclampsia is more prevalent in women under 20 or over 40 years of age. Those with pre-existing existing conditions of diabetes mellitus, renal diseases, high blood pressure, family history of preeclampsia, or previous complications with preeclampsia, are often at increased risk.
Although preeclampsia usually occurs during the second half of pregnancy, placental abnormalities related to preeclampsia appear early in the pregnancy. In normal pregnancies, a certain type of cells, known as cytotrophoblast stem cells, invade the uterus to help exchange nutrients and oxygen between the mother and the fetus. In preeclamptic patients, cytotrophoblast stem cells develop abnormally and invade into the placenta only shallowly. This shallow invasion prevents adequate blood flow to the placenta and deprives normal oxygen and food flow to the fetus. Babies born to preeclamptic women are often underweight due to inadequate nutrition and availability of oxygen.
Depending on the severity of the condition, preeclampsia varies from mild to severe. Mild preeclampsia is characterized by blood pressure readings of about 140/90 mm Hg, less than 5 g of protein in the urine a day, and swelling of face and hands. More severe forms of preeclampsia are characterized by blood pressure readings of about 160/110 mm Hg, over 5 g of protein in the urine a day, and beginning signs of end organ damage. Headaches, upper abdominal pains, impaired vision, fever, and vomiting are additional symptoms of preeclampsia. In extreme cases, preeclampsia can develop into eclampsia, which may lead to death and is characterized by seizure and coma.
Despite early treatment of mothers diagnosed with preeclampsia with aspirin and calcium, recent research has proven these treatment methods to be disappointing. To minimize risks to both mother and fetus, early detection is one of the most important factors in providing timely medical supervision and adequate expert care. In order to ensure that each case is detected at the earliest possible stage, it is also necessary to screen all mothers who are at risk for developing preeclampsia. Thus, a pressing need exists for an accurate, easy and cost effective method of detecting preeclampsia.
The present invention relates generally to methods for detecting preeclampsia in pregnancies. Specifically, the present invention comprises the steps of obtaining a sample specimen from a patient, assaying the specimen to determine the level of glycerophosphatidyl compounds, glycerophosphatidylcholine, lysophospholipids and/or lysophosphatidylcholine in the sample, comparing levels in the sample to levels in normal samples, and correlating significant changes as compared to normal samples as a positive indicator of preeclampsia.
In a preferred embodiment of the diagnostic method of the invention, the total amount of lysophospholipids (LPX) in a sample is measured by conversion into glycerol-3-phosphate (G3P). In an especially preferred embodiment, the sample specimen is incubated with lysophospholipase and a non-specific glycerophsphoryl compound phosphodiesterase to produce G3P from LPX. Then, the concentration of G3P thus produced is thereafter determined using an enzymatic cycling reaction.
In another preferred embodiment of the diagnostic method of the invention, the total amount of glycerophosphatidyl compounds (GPX) in a sample is measured by conversion into glycerol-3-phosphate (G3P). In an especially preferred embodiment, the sample specimen is incubated with a non-specific glycerophsphoryl compound phosphodiesterase to produce G3P from LPX. Then, the concentration of G3P thus produced is thereafter determined using an enzymatic cycling reaction.
In another preferred embodiment of the invention, the amount of lysophosphatidylcholine (LPC) in the sample is measured by enzymatically liberating choline from GPC. In especially preferred embodiments, samples are incubated with lysophospholipase and glycerophosphorylcholine phosphodiesterase to liberate choline from LPC. Choline is then preferably quantified using choline oxidase in a colorimetric reaction.
In another preferred embodiment of the invention, the amount of glycerophosphatidylcholine (GPC) in the sample is measured by enzymatically liberating choline from GPC. In especially preferred embodiments, samples are incubated with glycerophosphorylcholine phosphodiesterase to liberate choline from GPC. Choline is then preferably quantified using choline oxidase in a calorimetric reaction.
In yet another preferred embodiment of the invention, the amounts of at least two markers in the sample are measured, wherein the markers are chosen from the group consisting of glycerophosphatidyl compounds, glycerophosphatidylcholine, lysophospholipids, and lysophosphatidylcholine.
Another aspect of the present invention concerns diagnostic kits for the determination of preeclampsia in pregnant patients according to the above claimed method. Preferred embodiments of the diagnostic kits include enzymes and reagents necessary for the determination of the level of total glycerophosphatidyl compounds, glycerophosphatidylcholine, lysophospholipids and/or lysophosphatidylcholine in a specimen obtained from a pregnant patient, and instructions for making a preeclamptic diagnosis utilizing the kit. It is also preferred that the kit contain normal GPX, GPC, LPX or LPC level standards for comparison to the specimen obtained from the patient.